PROTON-PUMPING AND THE INTERNAL PH OF YEAST-CELLS, MEASURED WITH PYRANINE INTRODUCED BY ELECTROPORATION

The internal pH of yeast cells was determined by measuring the fluores cence changes of pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), w hich was introduced into the cells by electroporation. This may be a s uitable procedure for the following reasons. (i) Only minor changes in the physiological status of the cells seemed to be produced. (ii) The dye did not seem to leak at a significant rate from the cells. (iii) Different incubation conditions produced large fluorescence changes in the dye, which in general agree with present knowledge of the proton movements of the yeast cell under different conditions. (iv) Pyranine introduced by electroporation seemed to be located in the cytoplasm an d to; avoid the vacuole, and therefore it probably measured actual cyt oplasmic pH. (v) Correction factors to obtain a more precise estimatio n of the internal pH are not difficult to apply, and the procedure may be useful for other yeasts and microorganisms, as well as for the int roduction of other substances into cells. Values for the cytoplasmic p Hs of yeast cells that were higher than those reported previously were obtained, probably because this fluorescent indicator did not seem to penetrate into the cell vacuole.